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Larsson, L I; Sundler, F; Håkanson, R
doi: 10.1177/23.12.1104707pmid: 1104707
Treatment with formaldehyde gas and HCl vapor, simultaneously or in sequence, induces fluorescence with indoles, including tryptophan residues of peptides, as is evident from studies on protein droplet models. Among cells that display intense formaldehyde-HCl-induced fluorescence are pancreatic exocrine cells, gastric chief cells, Paneth cells and enterochromaffin cells. Peptide hormone-producing cells that can be visualized by the formaldehyde-HCl treatment include gastrin cells and glucagon cells. The simultaneous procedure has proved superior to the sequential procedure. Simultaneous formaldehyde-HCl treatment appears to be a useful method for the demonstration of tryptophan residues of peptides and proteins. It seems more sensitive than previously described indole methods.
doi: 10.1177/23.12.53248pmid: 53248
Theoretical considerations on the expected kinetics of the course of the Feulgen-Schiff reaction show that the leveling off of the first part of the Feulgen hydrolysis curve can be explained by the gradual conversion of deoxyribonucleic acid (DNA) to apurinic acid (APA). In addition, depolymerization of DNA caused by the acid used for hydrolysis can account for the decline after a maximum is reached in this curve. With the aid of polyacrylamide model films containing DNA, a detailed study was made both of the process of purine liberation which results in the formation of APA and of the depolymerization processes which cause losses of stainable material. The liberation of purine bases was analyzed by ultraviolet absorbance measurements and by gel chromatography of the neutralized hydrolysing acid. APA concentration was monitored by following the loss of ultraviolet absorbance associated with the purine losses. The depolymerization process was followed by phosphorus determinations. The experimental results were found to be in accordance with the kinetics expected from the theoretical model.
doi: 10.1177/23.12.53249pmid: 53249
As models for different states of chromatin compactness, nuclei from chicken erythrocytes were isolated and either osmotically swollen or kept as condensed as possible. Both types of nuclei were then fixed and incorporated into polyacrylamide films. Hydrolysis with 5 N HCl and staining with Schiff's reagent of these model films were studied using several parameters. The phosphate content of the films was analyzed as a parameter for the depolymerization losses and the staining with Schiff's reagent as a parameter for the apurinic acid (APA) content. The loss of ultraviolet absorbance from the films and the accumulation of ultraviolet absorbing substances in the hydrolyzing acid were monitored as parameters for the progress of hydrolysis. Conversion of the generated aldehyde groups to APA-Schiff chromophore is shown to take place with the same stoichiometry for both types of nuclei as well as for DNA in model films. It is further shown that the nuclei- and DNA-films are suitable models for investigating the influence of chromatin compactness on the course of the Feulgen-Schiff reaction. For the most compact form of chromatin studied, a very high reduction in staining intensity of up to 40% could be demonstrated after certain normally applied hydrolysis times. This is due primarily to a decrease with a factor of 2.3 of the depurination rate constants of these models (from 0.030/min to 0.013/min). Therefore prolonged hydrolysis periods are required to obtain the same APA concentrations, but then depolymerization processes cause losses of nuclear material. The differences in depurination rates could be explained by a decrease in [H3O]+ in the neighborhood of the purine-sugar linkages, caused by the presence of fixed positive charges form the protein components of the chromatin. These findings may explain the cytophotometrically determined differences in chromophore yield of 10-20% found in the nuclei of cells with different states of compactness of their chromatin. The descending part of the Feulgen hydrolysis curve represents the depolymerization of APA and loss by diffusion of the reaction products. In the Appendix, cytophotometric data of cells have been analyzed to show that this part of the hydrolysis curve may be used to estimate the acid stability of chromatin complexes. The depurination and depolymerization rates found closely correspond with the data obtained from the model films.
Boren, H G; Wright, E C; Harris, C C
doi: 10.1177/23.12.1194673pmid: 1194673
Emulsion sensitivity, latent image fading, and the effects of temperature, humidity, radiation dose and chemography on them were measured for NTB2 autoradiographic emulsion using quantitative methods. Sensitivity of NTB2 emulsion increased as the temperature during exposure increased, with the greatest increase per degree occurring between -20 degrees C. At 4 degrees C, emulsion sensitivity remained constant with time and radiation dose. Direct measurement of latent image fading showed no latent image fading for 60 weeks on slides exposed at 4 degrees C with Drierite. Slides exposed at 27 degrees C showed significant latent image fading and great variation between samples. High humidity decreased emulsion sensitivity and increased latent image fading. No evidence of either positive or negative chemography was found. The practical use of autoradiography requires an internal standard on each slide to correct for fluctuations in temperature and humidity during exposure time.
doi: 10.1177/23.12.172556pmid: 172556
A rabbit monospecific anti-rat uterus collagenase antibody has been used to study the distribution of collagenase in normal rat tissues by immunohistochemical methods. Indirect staining was performed with fluorescein-conjugated goat anti-rabbit immunoglobulin G antibody. The organs studied were brain, lung, myocardium, liver, spleen, kidney, adrenal, testes, uterus, xiphoid cartilage, tail tendon, skeletal (triceps) muscle and skin. Collagenase is widely present throughout the connective tissue structures in all organs examined. The enzyme is apparently bound to collagen fibers, reticulum fibers and basement membranes. The results suggest that control of collagenase activity depends on factors other than the presence of the enzyme in tissues.
doi: 10.1177/23.12.127811pmid: 127811
Tissues from mice were fixed in 1.5% glutaraldehyde, treated for the ultrastructural localization of alkaline phosphatase or Mg++-dependent adenosine triphosphatase, post-fixed in osmium tetroxide, dehydrated and embedded in plastic for electron microscopy. The sites of reaction were visualized in 1-mu plastic sections counterstained with toluidine blue, using a phase contrast microscope. The data show a close correlation between the sites of reaction observed with the phase contrast microscope and the sites studied with the electron microscope. The use of this technique for the study of these phosphatases in normal and pathologic tissues is recommended in order to achieve a high degree of accuracy in selecting a portion of the tissue sample for electron microscopy and to obtain greater resolution in the localization of these enzymes with the light microscope.
doi: 10.1177/23.12.1104708pmid: 1104708
The distribution of lysozyme (LZM) in normal human tissues was determined with the use of the immunoglobulin-enzyme (peroxidase) bridge method. LZM was detected in the following cells and tissues: secretory cells of the lacrimal gland, ductal epithelial cells of the parotid gland and the serous parts of the mixed sublingual glands, the esophageal submucosal glands, bronchial serous submucosal glands, gastric and pyloric glands, Brunner's glands of the duodenum, the Paneth cells of the small intestine, Kupffer cells of the liver and renal proximal tubular cells. In addition, LZM was also found in the mononuclear or polymorphonuclear cells of the placenta, lung, lamina propria of the small intestine, lymph nodes and spleen. This distribution of LZM is discussed in relation to its possible physiologic role in human tissues and particularly to its known antibacterial properties.
doi: 10.1177/23.12.53250pmid: 53250
For this study of photographic densitometry, sections of cartilage stained with Alcian Blue, safranin O and high iron diamine were photographed at x40 with Nikon photomicrography equipment on Kodak Panatomic X film with appropriate filters to enhance contrast. Portions of the developed negative films were selected from intercellular matrix regions, and circles of film equivalent in diameter to a 30-mu circle of tissue were obtained with a hand-held paper punch. Silver was eluted from the circles of film with 35% nitric acid, and the quantity of silver deposited on the film was determined by atomic absorption spectrophotometry as a measure of stain intensity. The intensity determined by this analytic procedure compared favorably with results obtained previously from the same tissue with microspectrophotometry. This method of silver analysis has advantages over earlier studies which used silver elution to determine photographic densitometry in its technical ease, accuracy and sensitivity. Furthermore, this method compares well with microspectrophotometry in its results and has the advantages of relative inexpensiveness and availability of equipment.
Takeuchi, T; Sasaki, M; Miyayama, H; Oyumi, M; Miyajima, H
doi: 10.1177/23.12.172557pmid: 172557
During the investigation of the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system in various tissue cells, it was observed that focal synthesis localized in a certain area of the cytoplasm occurred in some cells. This differed from the usual synthesis in which particles of similar size were synthesized within the cytoplasm. Otherwise, cytoplasmic particles of various size were also synthesized in other cells under the same histochemical condition. The possible significance of the presence of these patterns in glycogen synthesis is discussed.
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